There are great needs for cutting-edge technologies and systems that can analyze, isolate and quantify individual analytes within a heterogeneous mixture in a high-throughput format. These technologies may find broad applications in biology and chemistry that include pathogen detection, liquid biopsy, affinity reagent screening, rare cell enrichment, metabolite profiling, synthetic biology screening, and drug discovery. Conventional high-throughput systems are commonly based on a microwell plate that is often coupled with robotic liquid handling and flow cytometry systems. An advantage of these traditional systems is the capability to provide precise sample indexing because of the physically fixed well position. Nonetheless, these systems often require expensive instrumentation, large sample and reagent volume, yet with a relatively low throughput. More often than not, users either cannot afford the instruments, or, simply do not have enough starting samples available for the analysis, particularly in cases that involve precious samples such as biopsy, stem cells, and other rare samples.
Microfluidic-based high-throughput technologies are increasingly recognized as an effective alternative to the traditional microwell plate and robotic liquid-handling systems. These microfluidic technologies often partition a bulk sample into many isolated sub-nanoliter compartments in order to increase the effective analyte concentration and simultaneously reduce interferences from irrelevant species present in the same bulk sample. Droplet-based microfluidics represent a fast-growing technology that combines the ability of compartmentalization, the flexibility of fluidic droplet manipulation and the capacity for ultra-high throughput screening. It has additional advantages exemplified by minimal solution evaporation or adsorption of molecules at the device walls. Monodispersed (i.e., homogeneous size) droplets in the range of picoliter to nanoliter volume can be readily generated by a skilled person at kilo-hertz frequencies with sizes precisely controlled by fluid composition, flow rate and droplet-generation device geometry.
Droplet microfluidic devices have been developed for a wide range of applications including directed molecule evolution, pathogen detection, PCRs, single-cell and single-molecule analysis.
There are great needs for microfluidic droplet-based assays that allow precise indexing and tracking of individual assay droplets in a high throughput manner. One clear advantage with the droplet indexing is to enable kinetic or real-time detection of the assay signals, which may effectively identify a potential false-positive or false-negative reaction, and the ones with a user-defined biological or physicochemical property. In the context of drug screening or rare species isolation, there may be further needs to sort the identified droplets for a downstream application. To achieve the similar indexing capacity of a conventional microwell-plate system, there are a few prior designs of droplet-trapping structures or arrays in a microfluidic device. These location-fixed trapping structures may facilitate droplet indexing and detection in a kinetic manner. For instance, one approach used multiple channels with tandem constrictions to trap thousands of droplets, wherein the droplets were subsequently recovered by increasing the flow rate to push the select droplets through the channels. Similarly, another approach exploited the buoyancy of water-in-oil to trap thousands of droplets of sub-nanoliter size in an inverted floating array structure. The droplets in the inverted floating array can be recovered by flipping the device and flushing the trapping wells.
There are examples of stationary one- or two-dimensional droplet arrays. While these fixed-location array designs may enable multi-timepoint kinetic analysis of biological or chemical processes at a low throughput manner similar to that of a conventional plate system, these devices suffer a few critical drawbacks that make their real-world applications rather difficult and limited: 1) the manipulation and sorting of droplets from these types of structures are highly inefficient or virtually impossible; 2) detection synchronization across the droplets is hard to achieve for a large number of assay samples; 3) droplet trapping efficiency is often poor, where the majority of droplets simply flow through the device without being trapped and assayed, leading to significant loss of assay samples that may be precious and hard to procure; and 4) assay throughput is often limited, which is often below 10,000 and thus insufficient for important applications that involve the analysis and screening of a large number of analytes.
On the other hand, there are a few droplet-encoding methods in the context of biochemical assays. For example, there has been proposed for an alternating color-code to identify individual assay droplets with analytes. There is also report on the usage of varied concentrations of magnetic particles to encode assay droplets. Another approach uses a silicon-nanowire based field effect transistor as an electrochemical sensor to probe individual droplets on a grid. Nonetheless, droplet-encoding approaches represented by these prior arts present a few critical pitfalls: (1) the encoding material is commonly co-encapsulated with the assay sample in the same individual droplets, which may adversely affect the accuracy of the assay result and/or the integrity of intra-droplet sample that may be precious and intended to be recovered for further applications; (2) the available combinations of distinguishable codes are usually below 10,000, 1,000 or even 100, which unfortunately precludes any high throughput assay that requires a large amount of droplets in the scale of 104, 105, or higher; (3) complex or expensive instrument setups may be required, and/or (4) in some cases, the underlying detection-decoding process is slow (at the scale of a few tens of milliseconds), which may significantly limit the speed and robustness of the assay.
Thus, new methods and systems are greatly needed, which can precisely control droplet indexing and perform kinetic analysis of indexed droplets in an ultra-high throughput manner. Moreover, at least in the contexts of drug screening and rare cell isolation, there are additional unmet needs to couple the droplet indexing and detection with a downstream sorting module to efficiently recover assay droplets with a pre-defined kinetics property for further analysis or applications.